We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.DNA sequences engineered in vitro can be readily introduced into the genome of Saccharomyces cerevisiae cells by homologous recombination, facilitating the creation of duplications, insertions, and deletions of anything from single genes to large chromosomal fragments (33,38). In contrast, early experiments with mammalian cells showed that recombinant DNA molecules are most often inserted randomly into the genome by a mechanism that does not require extensive identity between the DNA fragment and the genomic sequences (32,40,47). More recently, it was shown that the efficiency of homologous gene replacement in mammalian cells can be greatly enhanced relative to random integration by using DNA sequences from a source that is isogenic to the recipient cells, suggesting that the presence of mismatches between the DNA fragment and the genome strongly inhibits homologous recombination (10, 45).DNA sequence mismatching presents a considerable barrier to homologous recombination in a wide variety of systems (5, 9, 10, 14, 21, 31, 34-36, 41, 45, 49, 50). Several laboratories have observed that defects in the mismatch repair machinery in bacterial species greatly lower the barrier against recombination between mismatched sequences (12,18,25,29,36,54). Other investigators have shown that mutations in mismatch repair genes in yeast (9, 34) and mammal (11) cells similarly reduce the inhibitory effect of mismatches, indicating that this genetic mechanism is evolutionarily conserved. It has been suggested that nonidentical sequences are prevented from recombining because the mispairing that occurs when heteroduplex DNA is formed is recognized by the mismatch repair machinery, after which the heteroduplex is unwound (9) or multiply nicked (27). The mismatch repair machinery in yeast corrects mismatches in the heteroduplex fo...

We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules.All organisms must repair the damage to their DNA that results from environmental stress and normal metabolism. Genetic recombination is one of several pathways that have evolved to repair this damage (13). This mode of repair can lead to deleterious consequences, however, because recombination between dispersed duplicate (ectopic) sequences can result in genome rearrangements or gene inactivation or both (42). These events are also implicated in human disease (20,24). Both DNA sequence length (2,26,51,53,58) and identity (6,18,36,43,45,47) are important determinants of the rate of recombination between repeated DNA sequences. What remains largely unclear is how short sequence length and mismatching hinder recombination. We took a genetic approach to studying the control of ectopic recombination in Saccharomyces cerevisiae. In a search for mutants that stimulate this recombination, we isolated an allele of the RAD3 gene (rad3-G595R) that increased the rate of recombination between sequences that share short lengths of perfect or imperfect homology.The RAD3 gene encodes a helicase (61) that is an essential component of the transcription and DNA repair complex TFIIH (factor B [10,66]) and is highly homologous to the human nucleotide excision repair gene XPD (60, 67). Many rad3 mutants have been isolated (13). These mutants exhibit a wide array of overlapping phenotypes including altered transcription (16), DNA repair (40,48,72), and recombination (37). One set of mutants, originally designated rem, were identified on the basis of their mutator and hyperrecombination phenotypes (14). The rem genes were subsequently found to be alleles of RAD3 and are thought to lead to the creation of recombinogenic double-strand breaks (37). Another mutant allele, rad3-2, is not hyperrecombinant but does decrease gene conversion tract length during intrachromosomal recombination (1). These diverse recombination phenotypes may be due to defects in the transcription of important recombination genes or to direct effects of mutant Rad3 proteins on recombination.White and Haber (71) analyzed the repair of double-strand breaks in DNA by homologous recombination between unlinked, duplicate sequences at the molecular level and showed that double-strand breaks are subject to extensive 5Ј-end degradation in wild-type cells. This processing is thought to be crucial for producing a single-stranded 3Ј end (3, 7...

Maintaining genome stability requires that recombination between repetitive sequences be avoided. Because short, repetitive sequences are the most abundant, recombination between sequences that are below a certain length are selectively restricted. Novel alleles of the RAD3 and SSL1 genes, which code for components of a basal transcription and UV-damage-repair complex in Saccharomyces cerevisiae, have been found to stimulate recombination between short, repeated sequences. In double mutants, these effects are suppressed, indicating that the RAD3 and SSL1 gene products work together in influencing genome stability. Genetic analysis indicates that this function is independent of UV-damage repair and mutation avoidance, supporting the notion that RAD3 and SSL1 together play a novel role in the maintenance of genome integrity.